Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.
Therapeutic Importance of G Protein-Coupled Receptors
Intercellular communication in multicellular organisms relies on numerous signal transduction pathways that allow chemical messages to be sensed extracellularly and converted into intracellular responses. One of the most ancient and well-diversified pathways uses G protein-coupled receptors (GPCRs) as the chemical sensor. GPCRs comprise a large family of transmembrane signaling proteins that are key to a variety of cellular activities including phototransduction, olfaction, neurotransmission, and endocrine function.
There are currently about 300 molecularly identified GPCRs and this number is rapidly growing. Estimates based on genomes that have been entirely sequenced suggest that there may be more than 1000 GPCRs in humans. The fact that a large proportion of prescribed drugs act on GPCRs coupled with the evidence of a large reserve of undiscovered genes suggests that these proteins will continue to be major targets for drug discovery for the foreseeable future.
Signaling Pathways Used by GPCRs
GPCRs mediate diverse cellular responses to external stimuli through their interaction with a single class of proteins known as heterotrimeric G proteins (G proteins). These proteins are composed functionally of two subunits, an α subunit that possesses GPCR-recognition and GTP-binding domains, and a dimer formed by β and γ subunits (Bourne, 1997; Lambright et al., 1996). Stimulated by agonist binding, GPCRs induce a conformational change in the G protein that facilitates the exchange of GDP for GTP bound to the α subunit. In the GTP-bound state, the α subunit is free to dissociate from the βγ dimer, permitting the two subunits to independently interact with a number of membrane-bound effector proteins including enzymes and ion channels.
To date, there are 17 Gα subunits that have been cloned (Simon et al., 1991). These fall broadly into four classes: those that activate phospholipase C (Gαq, Gα11, Gα14, Gα15, and Gα16), those that stimulate adenylate cyclase (Gαs and isoforms), those that mediate inhibition of adenylate cyclase and also permit interaction with a variety of other effectors through release of βγ subunits (Gαi and Gαo isoforms), and finally Gα12 and Gα13 whose regulatory functions are less well understood. By detecting and discriminating among structural features of both βγ and Gα, the individual GPCR activates only a subset of available G proteins (Bourne, 1997).
The “funneling” of signaling events through specific classes of G proteins has had important consequences for the design of assays to test the functional status of a given receptor. For example, receptors that couple strongly to Gαq, such as α1A-adrenoceptors, 5-HT2, receptors, or H1 histamine receptors, activate phospholipase C isoforms, initiating a rise in inositol phosphates (IP3) and a release of calcium from intracellular stores. Specific assays have been developed to measure the release of these signaling molecules. Likewise, other assays have been developed for measuring accumulation or depletion of cAMP (from stimulation or inhibition of adenylate cyclase) due to stimulation of receptors coupling either to Gαs or Gαi, respectively. A myriad of other assays have been elaborated that measure ion channel, GPTγS binding, MAP kinase, or transcriptional activities. In further elaborations of these methods, artificial “reporter genes” are used to provide a simplified endpoint initiated by some of the above cellular responses.
Ligand Identification for GPCR “Orphan” Receptors
The discovery of new GPCRs has outpaced the identification of new natural ligands, leading to a growing list of “orphan” G protein-coupled receptors whose ligand is unknown. Identifying the ligands for these orphan receptors is critical for determining their biological importance and will permit investigations into receptor pharmacology and drug design. While it is possible to identify ligands by binding, such assays depend upon the availability of high affinity radiolabeled ligands, and often on high levels of expression of the cloned receptor. On the other hand, functional activity can be elicited using unmodified, naturally occurring ligands applied to cells expressing moderate densities of receptor. The primary disadvantage of the functional approach is not knowing which class of G protein will couple efficiently to the orphan receptor. Although much progress has been made toward identifying motifs within the intracellular portions of GPCRs that bind G proteins, currently it is not possible to predict which class of G protein will couple to a given receptor. This uncertainty requires the employment of multiple functional assays for each orphan receptor in order to cover all possible signal transduction pathways. The availability of a single, genetically modified G protein that could couple universally to the vast majority of GPCRs would be an extremely useful tool for the study of orphan receptors and for the development of new therapeutic agents targeting GPCRs.
“Promiscuous” G Proteins and Modified G Proteins
The design of a universal functional assay for all GPCRs is a highly sought after goal for the pharmaceutical industry. Such an assay would eliminate the need to run multiple parallel assays for each receptor. Work on the Gα16 subunit (Offermans and Simon, 1995) showed that a single G protein can “route” receptors that normally couple to inhibition of adenylate cyclase to stimulation of inositol phosphate production (Offermanns and Simon, 1995). Such a system can take advantage of instrumentation that detects Ca++ mobilization via fluorescent dyes in a multiwell plate format suitable for mass screening of compound libraries. Unfortunately, while heterologous expression systems incorporating Gα16 are amenable to mass screening, there are a significant number of GPCRs that do not couple well to this G protein, reducing its general utility for screening orphan receptors.
Studies of the three dimensional structure of native G proteins (Lambright et al., 1996) and the functional activities of chimeric G proteins (see for review, Milligan and Rees, 1999) point to two regions of the Gα subunit that are involved in receptor recognition. Conklin and co-workers (Conklin et al., 1993) provided experimental evidence that the extreme C-terminal regions of Gαq, Gαs, and Gαi2 are important for directing targeting to the receptor. For example, replacing the last five amino acids of Gαq with the corresponding amino acids from Gαi2, permitted three receptors, which normally couple to Gαi/o, to stimulate phospholipase C (PLC). Similarly, replacing with the terminal five amino acids of Gαs, permitted stimulation of PLC by the vasopressin V2 receptor, which normally activates adenylate cyclase (Conklin et al., 1996). Other experiments, in which Gαs was altered by the C-terminal amino acids of Gαq, demonstrated the generality of the finding that a given G protein can be re-directed by replacing the C-terminus of a given Gα “backbone” with the appropriate C-terminus of another Gα subunit (see for review, Milligan and Rees, 1999). Thus, the C-terminus of Gα is one important determinant for GPCR recognition and may be modified to channel responses from the preferred signaling pathway to another one that would be amenable to automation.
The N-terminus of Gα is also involved in directing G protein to a target receptor, but the specificity for this is much less well understood. Kostenis and co-workers (Kostenis et al., 1997; Kostenis et al., 1998) noted that the N-termini of Gαq and Gα11 are unique in that they contain a six amino acid extension not found in other Gα subunits. Deletion of this extension permitted GPCRs that do not normally couple to wild-type Gαq, to productively couple to the mutant and activate PLC. Although N-terminal deletion mutants of Gαq improve coupling to Gαi/o-coupled receptors, the amplitude of second messenger response in many instances is low and not sufficient for mass screening applications.
Use of Ancestral G Proteins
Sequence analysis of Gα genes from organisms spanning multiple phyla suggests the existence of a primordial Gα ancestor (Wilkie and Yokoyama, 1994; Seack et al., 1998; Suga et al., 1999; FIG. 1). Lower organisms having less elaborate second messenger pathways and effector protein targets might harbor Gα homologues that are closer in structure to the ancestral protein. Further, these proteins may have the capacity to interact promiscuously with a wide variety of GPCRs because they lack structural motifs that subsequently evolved for the recognition of specific receptor subtypes. For example, in the search for primitive G proteins we noted that all invertebrate species, including Caenorhabditis elegans (C. elegans) and Drosophila melanogaster (D. melanogaster), lack the first six amino acids corresponding to the N-terminus of mammalian Gαq subunits. The use of Gα subunits from species that appear evolutionarily early on the phylogenetic tree offers an approach to universal coupling that has not been previously described.
C. elegans is an attractive organism because its genome has been completely sequenced (The C. elegans Sequencing Consortium, 1998) and because, as a pseudocoelomate, it branches early in the phylogenetic tree (Keeton, 1980). C. elegans contains only a single homologue from each of the four major Gα families: Gαq, Gαi, Gαs, and Gα12 (Jansen et al., 1999). This contrasts with mammals which have multiple isoforms within each of these families and, at the other phylogenetic extreme, yeast which has only two Gα subunits (Simon et al., 1991). The single Gαq subunit of C. elegans may, therefore, couple to a wider range of GPCRs than any of its mammalian homologues. When combined with specific C-terminal tails derived from mammalian non-Gαq subunits, the resulting chimeric G proteins may be further enhanced in their ability to efficiently couple to mammalian GPCRs.
This application describes the use of Gαq subunits obtained from invertebrate organisms, using C. elegans and D. melanogaster as examples, as “backbones” for the construction of chimeras. One chimera in particular, composed of C. elegans Gαq (cGαq) and modified to contain on its C-terminus the five amino acids of human Gαz (hGαz), exhibits surprisingly robust coupling to 78% of a large sample of cloned GPCRs. Further described are uses for this Gα chimera, and others, related to the identification of ligands for orphan GPCRs and for high-throughput screening of chemical compounds in functional assays.